PEI-somes and cationization of protein probes for microimaging of nervous system In this proposal we will develop a technology and a carrier for the delivery of fluorescently labeled fully functional antibodies into live cells. This will make possible immunohistochemical reactions inside live cells, eliminating the need for their fixation and processing. The objective is to advance microimaging of nervous system cells. Our approach will be based on a cationic polymer polyethyleneimine (PEI). We will develop a technology for the production of highly dispersed complexes of this cationic polymer with fluorescently-labeled antibodies, which will self-assemble into liquid particles with particle sizes below 200 nm (PEI-somes). These non-liposomal, non-micelle-based particles will be used for transmembrane delivery of functional antibodies. Application of PEI-somes as transmembrane carriers of fluorescently labeled antibodies for live cell immunohistochemistry will be developed into a marketable product for the needs of neuroscience and biomedical research. Although synthetic cationic polymers such as PEI are efficient DNA carriers, they were not used before as protein carriers. In our preliminary work we were able to assure strong complex formation and efficient delivery of functional proteins into cells. The specific aims of this project are: Aim 1: To develop the new enabling technology for the transmembrane transportation of proteins using their reversible cationization via formation of PEI-some particles. Demonstrate intracellular delivery of fluorescently labeled antibodies into live cells and their specific reactions with internal molecular targets, observable through two modalities - fluorescent microscopy and spectroscopy. To test the strength of the PEI-antibody complexes, their chemical properties and specific reactivity. Aim 2: To develop an application of cationized antibodies for the real-time microimaging of nervous system cells. To assess and optimize the effectiveness of transmembrane delivery of fluorescently tagged antibodies, their intracellular dynamics, toxicity and specific reactivity our preliminary data indicate superior qualities of the polymer-based protein carriers over the currently used peptide- and lipid-based protein delivery agents. Our project fills the gap in the field of protein transporters by developing a new antibody carrier. The particle-directed intracellular imaging will provide a technological advantage over currently used fixed-cell approaches. It will simplify detection and monitoring of intracellular processes and will also provide a method to monitor both toxicity and efficacy of treatment. The commercial potential for improved imaging agents is exceptional, as these agents are widely used in neuroscience and biomedical research. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of neuroscience and biomedical research. This technology will advance microimaging of nervous system cells and enable live-cell real-time imaging of cellular processes.